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Immune evasion of SARS-CoV-2 undermines current strategies tocounteract the pandemic, with the efficacy of therapeutic virus-neutralizing monoclonal antibodies (nAbs) being affected the most. In this work, we asked whether two previously identified human cross-neutralizing nAbs, iB14 (class VH1-58) and iB20 (class VH3-53/66), are capable of neutralizing the recently emerged Omicron (BA.1) variant. Both nAbs were found to bind the Omicron RBD with a nanomolar affinity, yet they displayed contrasting functional features. When tested against Omicron, the neutralizing activity of iB14 was reduced 50-fold, whereas iB20 displayed a surprising increase in activity. Thus, iB20 is a unique representative of the VH3-53/66-class of nAbs in terms of breadth of neutralization, which establishes it as a candidate for COVID-19 therapy and prophylactics.

A method for producing elemental boron nanoparticles with a size of less than 100 nm by ultrasonic dispersion in a liquid medium and subsequent cascade fractionation is described. The resulting boron nanoparticles were used as a target for boron neutron capture therapy (BNCT). According to the results of an experimental preclinical study of BNCT with the synthesized boron nanoparticles, neutron irradiation for 1 h of T98G human glioma cells pre-incubated in a medium with boron nanoparticles (10, 20, and 40 ppm in terms of boron-10 isotope) leads to a significant suppression of cell viability.

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We performed comprehensive profiling of humoral and B cell responses in a cohort of vaccinated subjects (n = 22), and demonstrate that Sputnik vaccination results in robust B cell immunity. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. 85 days after the first dose of the vaccine, ex vivo stimulated MBCs from the vast majority of Sputnik V vaccinees produced antibodies that robustly neutralized the Wuhan Spike-pseudotyped lentivirus. MBC-derived antibodies from all previously infected and some of the naïve vaccine recipients could also cross-neutralize Beta (B.1.351) variant of SARS-CoV-2. Virus-neutralizing activity of MBC-derived antibodies correlated well with that of the serum antibodies, suggesting the interplay between the MBC and long-lived plasma cell responses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. In vitro stimulated MBCs from previously infected recipients of Sputnik V secreted a significant amount of anti-RBD IgG both on days 28 and 85 from the beginning of vaccination. These antibodies demonstrated robust neutralization of the Wuhan Spike-pseudotyped lentivirus. In the naïve group of vaccinees, the level of anti-RBD IgG secretion was five- to six-fold reduced compared to that of the recovered group, and maximum virus neutralization (Wuhan spike) was achieved only on day 85. Sera from all the recovered and most naïve Sputnik V recipients were neutralizing against the ancestral Wuhan and mutant B.1.351 viruses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

Specific design features and main characteristics of a compact accelerator-based epithermal neutron source for boron neutron capture therapy are described. High quality of the neutron flux generated by the accelerator-based source has been experimentally confirmed. The opportunities for medical use of the accelerator-based epithermal neutron source in oncological centers for boron neutron capture therapy are assessed.

The adoptive transfer of allogeneic CAR NK cells holds great promise as an anticancer modality due to the relative ease of manufacturing and genetic modification of NK cells, which translates into affordable pricing. Compared to the pronounced efficacy of CAR T cell therapy in the treatment of B cell malignancies, rigorous clinical and preclinical assessment of the antitumor properties of CAR NK cells has been lagging behind. In this brief review, we summarize the biological features of NK cells that may help define the therapeutic niche of CAR NK cells as well as create more potent NK cell-based anticancer products. In addition, we compare T cells and NK cells as the carriers of CARs using the data of single-cell transcriptomic analysis.

Early results from clinical trials of autologous chimeric antigen receptor (CAR)-expressing T cells for the therapy of B-cell malignancies have encouraged extending the potency of this therapy to other cancers. However, the success of using CAR T-cells to treat patients with solid tumors has been limited. In this review, we summarize current knowledge on the design and applications of CARs for the targeted therapy of cancer. We describe existing issues that limit the widespread application of CAR T cells and discuss the optimization steps needed to further improve safety and efficacy of this therapeutic platform.

The article reports the development of a collection of lentiviral vector constructs enabling time-efficient production and testing of different variants of chimeric antigen receptors (CAR). These artificial surface proteins make it possible to redirect the activity of immune cytotoxic T-cells towards cancer cells. Chimeric antigen receptors usually encompass four functional modules, namely, antigen recognition, flexible linker, transmembrane, and signal modules. The use of modules with different properties allows modulating the affinity and specificity of CAR interaction with target antigens, as well as intensity and quality of activation signaling, which determines the cytotoxic properties of CAR T-cells, as well as their proliferation rate and time of persistence in the organism. The proposed vector system make it possible to easily test various combinations of CAR modules while its being open to distribution allows the direct comparison of the results obtained by different scientific groups.

Cloning

The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.

In the current article, we provide in vitro efficacy evaluation of a unique accelerator-based neutron source, constructed at the Budker Institute of Nuclear Physics (Novosibirsk, Russian Federation), for boron neutron capture therapy (BNCT), which is particularly effective in the case of invasive cancers. U251MG, CHO-K1 and V79 cells were incubated and irradiated in various concentrations of boric acid with epithermal neutrons for 2–3 h in a plexiglass phantom, using 2.0 MeV proton energy and 1.5–3.0 mA proton current, resulting in a neutron fluence of 2.16 × 1012 cm−2. The survival curves of cells loaded with boron were normalized to those irradiated without boron (to exclude the influence of the fast neutron and gamma dose components) and fit to the linear–quadratic (LQ) model. Colony formation assays showed the following cell survival rates (means ± SDs): CHO-K1: 0.348 ± 0.069 (10 ppm), 0.058 ± 0.017 (20 ppm), 0.018 ± 0.005 (40 ppm); V79: 0.476 ± 0.160 (10 ppm), 0.346 ± 0.053 (20 ppm), 0.078 ± 0.015 (40 ppm); and U251MG: 0.311 ± 0.061 (10 ppm), 0.131 ± 0.022 (20 ppm), 0.020 ± 0.010 (40 ppm). The difference between treated cells and controls was significant in all cases (P < 0.01) and confirmed that the neutron source and irradiation regimen were sufficient for control over cell colony formation. We believe our study will serve as a model for ongoing in vitro experiments on neutron capture therapy to advance in this area for further development of accelerator-based BNCT into the clinical phase.

The expression levels of the FCRL1 gene, which encodes a human B-cell surface receptor, were compared in healthy individuals and patients with autoimmune diseases. The expression levels were evaluated using DNA dot hybridization on membranes with spotted cDNA samples derived from blood-cell sub-populations of patients with autoimmune diseases. Quantification of the hybridization signals showed that FCRL1 expression in peripheral blood B-lymphocytes of patients with multiple sclerosis, lupus anticoagulants, Takayasu’s arteritis, and von Willebrand disease was significantly higher than in healthy individuals. Monoclonal and polyclonal FCRL1-specific antibodies that enable FCRL1 detection in Western blotting, immunohistochemistry, and flow-cytometry assays were generated. It was found that FCRL1 is expressed on the surfaces of mature CD19+ B-cells. In the tonsils, FCRL1-positive cells were located in the crypt area, i.e., in the mantle zone of secondary lymphoid follicles and among the cells of lymphoid epithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63 kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.

We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

In primates and rodents, the extended FcR family is comprised of three subsets: classical FcRs, structurally diverse cell surface receptors currently designated FCRL1-FCRL6, and intracellular proteins FCRLA and FCRLB. Using bioinformatic analysis, we revealed the FcR-like genes of the same three subsets in the genome of dog, another representative of placental mammals, and in the genome of short-tailed opossum, a representative of marsupials. In contrast, a single FcR-like gene was found in the current version of the chicken genome. This in silico finding was confirmed by the gene cloning and subsequent Southern blot hybridization. The chicken FCRL gene encodes a cell surface receptor with the extracellular region composed of four Ig-like domains of the D1-, D2-, D3-, and D4-subtypes. The gene is expressed in lymphoid and non-lymphoid tissues. Phylogenetic analysis of the mammalian and chicken genes suggested that classical FcRs, FCRLA, and FCRLB emerged after the mammalian-avian split but before the eutherian-marsupial radiation. The data obtained show that the repertoire of the classical FcRs and surface FcR-like proteins in mammalian species was shaped by an extensive recombination process, which resulted in domain shuffling and species-specific gain and loss of distinct exons or entire genes.

FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B-cell lymphoma, diffuse large B-cell lymphoma and lymphocyte predominance Hodgkin's disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut-off that distinguished subsets of diffuse large B-cell lymphoma. Our results also showed that FCRL tended to be negative in T-cell-rich B-cell lymphoma and in classical Hodgkin's disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B-cell neoplasms.

The genes encoding FcRgamma and TCRzeta homologs were identified using a bioinformatic approach in the amphibian Xenopus laevis. Deduced amino acid sequence of Xenopus TCRzeta is highly similar to the mammalian and avian counterparts, whereas that of FcRgamma differs by the presence of an additional ITAM-like motif. The presence of the negatively charged residue in the transmembrane regions of both subunits suggests their ability to serve as signal transducing modules in complex with activating receptors. The short extracellular regions contain characteristic cysteine residues responsible for dimerization in the mammalian subunits. According to Southern blot analysis, Xenopus laevis may possess two non-allelic genes for each subunit. Northern blots revealed FcRgamma transcripts of two sizes differentially expressed in thymus, spleen, intestine, liver and kidney. TCRzeta mRNA was predominantly expressed in the thymus and spleen. These data indicate that the amphibian immune system employs activating receptor complexes arranged in a mammalian-like way.

SLAMF9 is a member of the signaling lymphocyte-activating molecule (SLAM) immunoreceptor family. The SLAM family receptors are expressed in a broad range of immune cells and play an important role in immunity. To date, SLAMF9 is the least studied member of this family. Its ligand, signaling properties, and cells on whose surface it is expressed are unknown. We generated hybridoma clones 6E11 and 7G5 secreting monoclonal antibodies specific to human SLAMF9. BALB/c mice were immunized with Escherichia coli-expressed purified SLAMF9 protein; splenocytes from these mice were fused with mouse myeloma cell line NS-1. Based on isotyping of the MAbs, clone 6E11 was referred to the IgG1 subclass, while 7G5 to IgG2b. The specificity of these MAbs was assessed by ELISA, immunoblotting, immunohistochemistry, and flow cytometry. According to the results of epitope analysis, clone 6E11 reacts with the C2-like domain, whereas 7G5 is specific to the V-like domain of the SLAMF9 molecule. The generated MAbs were demonstrated to be applicable in various immunochemical analyses. They may be useful tools in studies clarifying the expression and function of human SLAMF9.

A novel conserved member of the leukocyte Fc receptor (FcR) family was identified in human and mouse. The presumably secreted protein, designated FCRL (FcR-like) is comprised of four domains. The three N-terminal domains are related to the extracellular region of FcγRI, with the second (35–37% residue identity) and the third (46–52%) domains showing highest similarity. The C-terminaldomain is a unique sequence enriched with proline residues. In humans, alternative transcripts for six FCRL isoforms were revealed. Spleen and tonsils were found to be the major sources of FCRL mRNA in human tissues. Western blotting of tonsil cell lysate using FCRL-specific antibodies recognized a 44-kDa protein produced as a monomer containing free sulfhydryl groups. The monomer, however, was able to form disulfide-linked homo-oligomer upon oxidation. In COS-7 cells transiently transfected with two human FCRL isoforms, both resided intracellularly. Immunohistochemical staining of tonsil sections demonstrated the FCRL expression in germinal centers, suggesting that the protein may be implicated in germinal center-specific stages of B cell development. The phylogenetic analysis of the FCRL relationships with the leukocyte FcR supports a view that the three-domain structure was primordial in the evolution of the family.

Subtractive hybridization was used to study river lamprey ( Lampetra fluviatilis ) leukocyte-specific cDNA. A clone representing the most abundant component (12 %) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3 ′ untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA-1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA-1 has the highest similarity to the chicken EMF-1 (40 %) and to the mammalian IL-8 (32 – 33 %). However, it lacks the ELR motif essential for the function of the mammalian IL-8-related chemokines. Based on the phylogenetic analysis of the LFCA-1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.

FCRLA is an ER-resident B-cell specific protein. The exact function of this protein remains unclear although human FCRLA has been recently shown to interact with IgM, IgG and IgA. The retention of FCRLA in ER is mediated by the N-terminal domain. The major human FCRLA isoform is encoded by five exons, of which one encodes a short signal peptide (SSP) and the others code four protein domains. Here we show that human tissues also produce transcripts which contain an additional exon and encode proteins with signal peptide that is six residues longer (LSP). Transfection experiments demonstrated that the extension of the signal peptide had no visible effect on the topology and molecular mass of the processed four-domain FCRLA isoform. However, the length of the signal peptide was found to affect processing of two-domain FCRLA isoforms composed of the third and fourth domains (FCRLAd2). The signal peptide was not cleaved in the SSP-FCRLAd2 and this isoform was found to accumulate in the ER. In contrast, the LSP-containing FCRLAd2 isoform was processed, O-glycosylated and secreted. The secreted FCRLAd2 isoform did not interact with IgG- or IgM-immunosorbents.

Screening of a mink cDNA library with an antibody probe resulted in the isolation of clone pIGL-2 containing an Ig lambda chain coding sequence. The sequence comprised almost the entire V segment as well as J, C, and 3'-untranslated sequences. A second clone, pIGL-10, was isolated by rescreening the cDNA library with the use of pIGL-2 as a probe. pIGL-10 was found to contain a frameshift deletion of a single nucleotide in the C region. pIGL-2 and pIGL-10 were 81% homologous to each other in the FR3 of the V segment, and 95% of homology was found in their C regions. The J segments of the two clones differed in only one nucleotide position. Comparison of cloned lambda chain sequences with those of other mammals revealed that mink V lambda and C lambda genes have the highest homology with their human counterparts. The V lambda sequence of clone pIGL-2 appears to be a homologue of human subgroup III V lambda genes. Southern blot hybridization of mink DNA with the C lambda and V lambda probes derived from pIGL-2 revealed five or six hybridizing C lambda fragments and at least 11 hybridizing V lambda fragments. This suggested that the lambda genes in carnivores, like those in primates, have duplicated extensively during evolution.

The ratio of kappa and lambda chains of immunoglobulins varies significantly from one species to another. It has previously been thought that lambda was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent lambda and kappa types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK-1 containing kappa chain-encoding sequence. The cDNA insert of pIGK-1 included most of the V segment, as well as the J, C and 3' untranslated sequences. Mink V kappa sequence shown the highest homology with the human V kappa II subgroup genes (76-79%). Mink C kappa sequence was 53-63% homologous to C kappa of other species. The striking feature of mink C kappa chain is the presence of glutamine in the C-terminal position. Southern blot analysis suggested that mink haploid genome has one C kappa gene and multiple V kappa genes. The kappa:lambda chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the kappa- and lambda-producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the lambda chains, thereby confirming that lambda chains in this species are additionally subdivided into several subtypes.

We studied the evolution of the CD2 family in tetrapods by extracting and analyzing CD2-like genes from the genome of the amphibian species Silurana (Xenopus) tropicalis. An exhaustive analysis of the genomic and cDNA databases resulted in the identification of at least 70 CD2-like genes. The predicted receptors mostly maintain the typical VC2 ectodomains, but are highly diverse in their C-termini, which suggests a broad range of signaling capacities. Apart from the presumed monomeric receptors with ITSM and/or ITIM motifs, the Silurana family includes secreted proteins. Furthermore, a fraction of the receptors contain a conserved TM subtype with the NxxR motif that is known to promote an association with the FcRγ subunit and that was previously found in the members of the FcR- and KIR-related receptors. The expression analysis of a sample of the genes showed broad tissue distribution and gene-specific expression patterns. Phylogenetic analysis predicted that the CD58, CD150/SLAM, and SLAMF8 genes were maintained as single-copy genes in both mammals and amphibians, while others expanded/contracted in a lineage-specific manner.

In this study, we searched the amphibian species Xenopus laevis and Silurana ( Xenopus ) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis . ILRs encode type I transmembrane receptors with 3–4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRγ subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

Receptors subdivided into inhibitory and activating forms play important roles in the regulation of leukocyte development and effector functions. Two prototypic examples of paired receptors are Fc-receptors (FcR) and Killer cell Immunoglobulin-like receptors (KIR). FcRs are cell surface proteins that bind to the constant regions of IgG and IgE. Classical KIRs recognize MHC class I molecules and regulate natural killer (NK) cell cytotoxic functions. The evolution of these proteins and the time of their origin remain enigmatic. So far, molecules unequivocally related to mammalian FcRs and KIRs have been identified in chicken and an amphibian Xenopus. The lineage-specific evolution of the FcR and KIR families apparently led to the generation of unique sets of receptors in all species studied. Members of both families show extraordinary diversity of domain architectures. This structural diversity makes elusive the functional relationships between the highly specialized mammalian FcR and KIR genes and their homologs in nonmammalian species.

BackgroundRecent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representativesXenopus tropicalisandXenopus laevis.ResultsThe diploid genome ofX. tropicaliscontains at least 75 genes encoding paired FcR-related receptors designated XFLs. The allotetraploidX. laevisdisplays many similar genes primarily expressed in lymphoid tissues. Up to 35 domain architectures generated by combinatorial joining of six Ig-domain subtypes and two subtypes of the transmembrane regions were found in XFLs. None of these variants are shared by FcR-related proteins from other studied species. Putative activating XFLs associate with the FcRγ subunit, and their transmembrane domains are highly similar to those of activating mammalian KIR-related receptors. This argues in favor of a common origin for the FcR and the KIR families. Phylogenetic analysis shows that the entire repertoires of theXenopusand mammalian FcR-related proteins have emerged after the amphibian-amniotes split.ConclusionFcR- and KIR-related receptors evolved through continual species-specific diversification, most likely by extensive domain shuffling and birth-and-death processes. This mode of evolution raises the possibility that the ancestral function of these paired receptors was a direct interaction with pathogens and that many physiological functions found in the mammalian receptors were secondary acquisitions or specializations.

FCRLA is a recently identified intracellular protein structurally related to the classic Fc receptors and expressed primarily in the germinal centers of B cells. We generated six monoclonal antibodies (MAbs) specific to the human protein. The MAbs recognize three different epitopes, which were shown to be localized on the D3 domain of the FCRLA molecule. The clones M101 and M616 were demonstrated to be applicable in various immunochemical analyses, such as immunoblotting, immunohistochemistry, and immunoprecipitation. In addition, this pair of antibodies was used for development of a sandwich version of ELISA to quantitatively detect FCRLA in cell lysates. Using these MAbs, we studied FCRLA expression in a panel of human B cell lines, such as Raji, Daudi, Bjab, BL-2, RPMI 1788, RPMI 8226, IM-9, and SKW6.4. It was found that all these lines, except RPMI 8226, produce FCRLA but may vary in the proportion of FCRLA-positive cells. The MAbs we established can be a useful tool to investigate the functional role of FCRLA and its applicability as a B cell development and malignant transformation marker.

We report cloning and characterization of FCRL2, a novel human gene that belongs to the FcR family. The gene is closely linked and structurally similar to the recently identified FCRL/FREB/FcRX gene. The encoded protein is composed of three Ig-like domains and a C-terminal mucin-like domain containing a conserved alpha-helical motif with dileucine signals. Intraexonic splicing may generate two alternative transcripts, coding for isoforms with the third and fourth domains replaced by entirely different amino acid sequences. Like FCRL, the full-length isoform of FCRL2 is expressed intracellularly in transfected 293T cells. Expression analysis revealed FCRL2 mRNA only in placenta. The gene transcripts were not detected in lymphoid tissues or in the main leukocyte subsets isolated from peripheral blood. However, we found that FCRL2 is differentially expressed by transformed B cell lines. Of interest is also the finding that the gene expression may be up-regulated in the progression of melanocytic tumors.

FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B-cell lymphoma, diffuse large B-cell lymphoma and lymphocyte predominance Hodgkin's disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut-off that distinguished subsets of diffuse large B-cell lymphoma. Our results also showed that FCRL tended to be negative in T-cell-rich B-cell lymphoma and in classical Hodgkin's disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B-cell neoplasms.

The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3epsilon, CD3gamma/delta, CD79a, CD79b, TCRzeta, FcRgamma, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost-tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.

It is believed that mouse Fc gamma RIII arose by an evolutionarily recent recombination, which brought together the extracellular domains from Fc gamma RII with the transmembrane/cytoplasmic region from the ancestor Fc gamma RIII. Here, we report identification of a mouse gene encoding a transmembrane receptor that may be regarded as the true ortholog of nonrodent CD16/Fc gamma RIII. Designated CD16-2, the novel protein is highly similar to human Fc gamma RIIIA in the signal peptide (60% identical residues), and in the extracellular domains (65%). Although the similarity between the two proteins is less conspicuous in the transmembrane/cytoplasmic region (54%), it is higher than between human Fc gamma RIIIA and mouse Fc gamma RIII (44%). However, the conserved transmembrane motif LFAVDTGL shared by rodent and human Fc gamma RIII and Fc epsilon RI has two replacements in CD16-2. The CD16-2 gene is tightly linked to the Fc gamma RIII and Fc gamma RII genes and consists of five exons. Northern blot analysis revealed that CD16-2 is expressed in peripheral blood leukocytes, as well as in spleen, thymus, colon and intestine. RT-PCR showed prominent expression in macrophage cell line J774. Based on sequence comparisons, it is suggested that the modern repertoire of the mammalian low affinity Fc receptors has resulted from repetitive duplications and/or recombinations of three ancestral genes.

A novel conserved member of the leukocyte Fc receptor (FcR) family was identified in human and mouse. The presumably secreted protein, designated FCRL (FcR-like) is comprised of four domains. The three N-terminal domains are related to the extracellular region of FcgammaRI, with the second (35-37% residue identity) and the third (46-52%) domains showing highest similarity. The C-terminal domain is a unique sequence enriched with proline residues. In humans, alternative transcripts for six FCRL isoforms were revealed. Spleen and tonsils were found to be the major sources of FCRL mRNA in human tissues. Western blotting of tonsil cell lysate using FCRL-specific antibodies recognized a 44-kDa protein produced as a monomer containing free sulfhydryl groups. The monomer, however, was able to form disulfide-linked homo-oligomer upon oxidation. In COS-7 cells transiently transfected with two human FCRL isoforms, both resided intracellularly. Immunohistochemical staining of tonsil sections demonstrated the FCRL expression in germinal centers, suggesting that the protein may be implicated in germinal center-specific stages of B cell development. The phylogenetic analysis of the FCRL relationships with the leukocyte FcR supports a view that the three-domain structure was primordial in the evolution of the family.

A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger receptor superfamily-related domain at the C-terminus. The other cDNAs code for the type I transmembrane proteins with the extracellular parts comprised of one to six Ig-like domains. Five homologous types of the Ig-like domains were defined and each protein was found to have a unique combination of the domain types. The cytoplasmic tails of the transmembrane proteins show different patterns of the tyrosine-based signal motifs. While the human IFGP members appear to be B-cell antigens, the mouse genes have a broader tissue distribution with predominant expression in brain. Sequence comparisons revealed that the IFGP family may be regarded as a phylogenetic link joining the leukocyte FcRs with the rat NK cell-specific gp42 antigen and platelet endothelial cell adhesion molecule-1 (PECAM-1), two mammalian leukocyte receptors whose close relatives were not found previously. It is suggested that FcRs, the IFGP proteins and gp42 have arisen by a series of duplications from a common ancestor receptor comprised of five Ig-like domains. The organization of the human genes shows that the IFGP family evolved through differential gain and loss of exons due to recombination and/or mutation accumulation in the duplicated copies.

CD3epsilon is an essential component of the T-cell receptor (TCR) complex for antigen. We report here molecular cloning and characterization of cDNAs encoding the CD3epsilon homologues in sterlet (Acipenser ruthenus), a representative of primitive chondrostean fishes. Sequence analysis of the cDNA clones demonstrated unexpectedly high CD3epsilon gene heterogeneity in this species. While some cDNAs encoded proteins with the structure typical of mammalian CD3epsilon, others coded for proteins lacking the membrane-proximal half of the extracellular domain. Two cDNAs contained in-frame stop codons in the region encoding the cytoplasmic domain. Based on genomic blot analysis and RT-PCR typing of individual spleen RNAs, we suggest that sterlet may possess two highly polymorphic CD3epsilon loci, of which one can produce alternatively spliced transcripts. The structural elements shown to be functionally important in the mammalian CD3epsilon are strongly conserved in the sterlet CD3epsilon. The cytoplasmic region contains an immunoreceptor tyrosine-based activation motif (ITAM) with YEPI and YSGL tyrosine-containing sequences that are characteristic of only this TCR subunit. The pattern of sequence conservation indicates also that strong selection pressure was imposed on a motif VYYW at the C-end of the transmembrane domain and on a CD3epsilon-specific proline-rich motif RXPPVP juxtaposed to the N-terminus of the ITAM. Weak similarity of the sterlet CD3epsilon with the chicken and Xenopus CD3gamma/delta indicates that these two TCR subunits diverged before radiation of bony fishes and tetrapods. While the role of CD3epsilon heterogeneity in sterlet remains to be elucidated, the data obtained show that the basic mechanisms of TCR signaling have ancient evolutionary origin.

The chemokine receptor CXCR4 and its ligand SDF-1 are essential components of hematopoiesis, organogenesis and immunomodulation in mammalian species. We cloned a cDNA encoding CXCR4 homolog of sterlet ( Acipenser ruthenus ), a representative of chondrostean fishes. The deduced amino acid sequence of sterlet CXCR4 is almost equally distant from mammalian and teleost CXCR4 (66–68% identical residues). Percent identity with the other chemokine receptors varies in the 30–35% range. The CXCR4 sequences from the three phylogenetically diverged lineages were compared with the sequences of the other chemokine receptors to determine the CXCR4-specific structural elements that were conserved during vertebrate evolution. The characteristic residues and/or motifs are located predominantly in the intracellular and extracellular regions and in the third, fourth and fifth transmembrane domains. The data presented may be helpful for structure-function analysis of the CXCR4 ligand binding and signal transduction.

Two cDNA clones, encoding mink Ig γ chains were characterized. The pIGG47 clone contains a part of the leader segment, VDJ and C regions, and pIGG14 contains a part of the J and a complete C region. The clones differ by only four nucleotides in the C region, and they most probably represent allelic variants of the same gene. The V gene segment of pIGG47 was found to be highly similar to human VHIII subgroup sequences; there was 86–87% similarity for the whole V gene segment and 91% for the VHIII specific regions (codons 65–87). Southern blot analysis demonstrated that a high proportion of mink VH genes is VHIII related. The V gene segment used as a probe revealed 19–23 bands in mink DNA under stringent conditions. This is in agreement with our previous data showing that a high proportion of mink Ig contains an ‘alternative’ binding site for protein A, a feature common to VHIII-related molecules. According to Southern blot analysis there may be 5–7 Cγ genes at the mink IgH locus.

A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).